The KpnI restriction endonuclease is an orthodox Type IIP enzyme, which binds to DNA in the absence of metal ions and cleaves in the presence of Mg2+. KpnI restriction endonuclease from Klebsiella pneumoniae, was characterized by Tomassini et al. Nucleic Acids Res. 5:4055-4064 (1978); Kiss et al. Nucleic Acids Res. 19:3460 (1991); Chandrashekaran et al. Nucleic Acids Res. 32:3148-3155 (2004) and cloned and sequenced as described in U.S. Pat. Nos. 5,192,675 and 5,082,784. KpnI is sold commercially in a buffer containing 10 mM Bis Tris Propane-HCl, 10 mM MgCl2, 1 mM dithtiothreitol (pH 7.0 at 25° C.) (New England Biolabs, Inc., Ipswich, Mass.).
KpnI recognizes the palindromic double-stranded hexameric DNA sequence 5′-GGTACC-3′ cleaving the DNA at the indicated position [] to generate a 3′, 4-base overhang (Kosinski et al. Proteins 53:369-379 (2003)). KpnI restriction endonuclease forms a restriction-modification system together with KpnI methyltransferase, which transfers a methyl group from the cofactor AdoMet onto the N6-position of the adenine in both strands within the same sequence.